Devices and methods that can accurately analyze analyte(s) of interest in a sample are essential for diagnostics, such as for example diagnosing a disease, disorder or condition, prognostics, environmental assessment, food safety, detection of chemical or biological warfare agents and the like. Most current techniques for quantifying low levels of analyte molecules in a sample use amplification procedures to increase the number of reporter molecules to provide a measurable signal. Examples of current techniques include enzyme-linked immunosorbent assays (ELISA) for amplifying the signal in antibody-based assays, as well as the polymerase chain reaction (PCR) for amplifying target DNA strands in DNA-based assays. Most detection schemes require the presence of a large number of molecules in the ensemble for the aggregate signal to be above the detection threshold. This requirement limits the sensitivity of most detection techniques and the dynamic range (i.e., the range of concentrations that can be detected). Many of the known methods and techniques are further plagued with problems of non-specific binding, which leads to an increase in the background signal and limits the lowest concentration that may be accurately or reproducibly detected.
Digital ELISA is a candidate for next generation of immunoassay as it can detect one molecule of enzyme using a conjugate. See FIGS. 1 and 2. In digital ELISA, target molecules are captured on beads between a capture antibody and a detection antibody, wherein the detection antibody is bound to an enzyme. The beads are then entrapped in a droplet chamber with the substrate of the enzyme, and the aqueous phase is displaced by a heavy oil, allowing removal of the aqueous phase before analysis.
In bead-based digital ELISA, single beads are encapsulated in microchambers of the array. Some of the chambers in which a bead has captured an immune-complex species provide bright spots upon fluorescence imaging (i.e., a bright chamber). The percentage of chambers having beads present is correlated to concentration of antigen. Therefore, it is necessary to identify positions of beads and enzymes in the microchambers of the array.
Optical imaging is a method to determine the location of beads in an assay. Optical imaging can also detect data for a large number (over 10,000) of microchambers at a time using bead/enzyme dual channel imaging. However, conventional optical systems for dual channel imaging are large and expensive to mount on existing products because of the complicated optics for fluorescence/fluorescence type of bead/enzyme dual channel imaging. Conventional optical systems require multiple optical filters for the respective channels, and an external actuator for exchanging the modes.